Methods and Compositions for the Treatment of Autoimmune and Inflammatory Diseases

ABSTRACT

Compositions and methods for the treatment of autoimmune and inflammatory diseases are disclosed.

This application is a continuation of U.S. patent application Ser. No.15/841,416, filed Dec. 14, 2017, which is a divisional of U.S. patentapplication Ser. No. 14/237,706, filed Apr. 23, 2014, now U.S. Pat. No.9,879,092, issued Jan. 30, 2018, which is a § 371 application ofPCT/US2012/050146, filed Aug. 9, 2012, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application No. 61/522,009,filed Aug. 10, 2011. The foregoing applications are incorporated byreference herein.

FIELD OF THE INVENTION

This invention relates generally to the field of autoimmune andinflammatory diseases. Specifically, the invention provides novelcompositions and methods for the treatment of diseases that have anautoimmune and/or inflammatory component in their pathology.

BACKGROUND OF THE INVENTION

Autoimmune disease occurs when an organism fails to recognize its ownconstituent parts as “self,” thereby resulting in an immune responseagainst its own cells and tissues. In other words, the body actuallyattacks its own cells. The immune system mistakes some part of the bodyas a pathogen and attacks it. Current treatments for autoimmune diseasestypically include immunosuppression and/or symptomatic treatment withnon-disease modifying anti-inflammatories in order to decreases thedamage of the aberrant immune response. However, there is a need in theart for methods and compositions for inhibiting and/or delaying theonset of pathology associated with autoimmune disorders.

SUMMARY OF THE INVENTION

In accordance with one aspect of the instant invention, methods forinhibiting, treating, and/or preventing the onset of an autoimmuneand/or inflammatory disease and/or diseases that have an autoimmuneand/or inflammatory component in their pathology in patients in needthereof are provided. The methods comprise the administration of atleast one RhoB inhibitor. In a particular embodiment, the RhoB inhibitoris an antibody or antibody fragment immunologically specific for RhoB ora peptide fragment thereof. In a particular embodiment, the RhoBinhibitor is a structurally related or derived small molecule of theantibody, antibody fragment, peptide fragment or chemical orbiologically mimetic of the antibodies' CDR regions and epitopesrecognized by the CDRs. In a particular embodiment, the RhoB inhibitoris a RhoB peptide. In a particular embodiment, the methods comprise theadministration of a composition comprising at least one RhoB peptideand/or antibody or antibody fragment immunologically specific for RhoBor a peptide fragment thereof and at least one pharmaceuticallyacceptable carrier. In a particular embodiment, the methods furthercomprise the administration of at least one anti-inflammatory agentand/or immunosuppressant concurrently and/or sequentially with the atleast one RhoB inhibitor (e.g., an antibody or antibody fragmentimmunologically specific for RhoB or a peptide fragment thereof).

Compositions for the inhibition, treatment, and/or prevention ofinflammatory or autoimmune disease are also provided. The compositionscomprise at least one RhoB inhibitor and at least one pharmaceuticallyacceptable carrier. In a particular embodiment, the RhoB inhibitor isantibody or antibody fragment immunologically specific for RhoB or apeptide fragment thereof. In a particular embodiment, the RhoB inhibitoris a RhoB peptide. In another embodiment, the composition furthercomprises at least one anti-inflammatory compound and/or at least oneimmunosuppressive agent.

The instant invention also provides anti-RhoB antibodies, RhoB peptide(e.g., for the generation of antibodies), or structurally related orderived small molecules of the antibody, antibody fragment, peptidefragment or chemical or biologically mimetic of the antibodies' CDRregions and epitopes recognized by the CDRs, and compositions comprisingthe same.

In accordance with one aspect of the instant invention, methods forinhibiting, treating, and/or preventing a condition or disorderassociated with increased levels of immunoglobulin in the blood serum(e.g., hypergammaglobulinemia or monoclonal gammopathy of undeterminedsignificance) in patients in need thereof are provided. The methodscomprise the administration of at least one RhoB inhibitor as describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the mean ankle thickness over time of K/BxN micewhich were treated with anti-RhoB-peptide serum, anti-KLH serum, orcarrier alone.

FIG. 2A is a graph of the titer of serum anti-glucose-6-phosphateisomerase (GPI) Ig from K/BxN mice treated with anti-RhoB-peptide serum,anti-KLH serum, or carrier alone. FIG. 2B is a graph of the number ofanti-GPI secreting cells per 10⁵ cells of K/BxN mice treated withanti-RhoB-peptide serum, anti-KLH serum, or carrier alone.

FIG. 3 provides the amino acid sequence of human RhoB (SEQ ID NO: 3).Underlined sequence is Peptide 1 (SEQ ID NO: 1).

FIG. 4 provides a graph of the IgM secretion with or withoutlipopolysaccharide (LPS) stimulation in the presence or absence of acontrol antibody or anti-RhoB antibodies from a hybridoma or a subclonethereof.

FIG. 5 provides a sequence alignment of RhoA (SEQ ID NO: 4) and RhoB(SEQ ID NO: 3). The underlined sequences and the boxed sequencesrepresent antigens for RhoB antibodies.

FIG. 6A provides a graph of rear ankle thickness±SEM of K/BxN micetreated with anti-RhoB monoclonal antibody 9G5 or 7F7 or control Igbefore the onset of arthritis (21 days of age). FIGS. 6B and 6C providegraphs of the anti-GPI autoantibody titers and the number of anti-GPIantibody secreting cells (ASCs) in the mice, respectively.

FIG. 7A provides a graph of the rear ankle thickness±SEM of K/BxN miceover a long time course that were treated with anti-RhoB monoclonalantibody 9G5 or control Ig after the onset of arthritis at 4 weeks ofage. FIG. 7B provides a graph of the rear ankle thickness±SEM of K/BxNmice over a shorter time course that were treated with anti-RhoBmonoclonal antibody 9G5 or 7F7 or control Ig after the onset ofarthritis at 4 weeks of age.

FIG. 8A provides a graph of rear ankle thickness±SEM in arthritic RhoBKO mice (RhoB KO KRN.g7). FIG. 8B provides a graph of rear anklethickness±SEM in naïve C57BL/6 mice that received a serum transfer fromKRN B6.g7 or RhoB KO KRN B6.g7 mice on day 0. FIG. 8C provides a graphof rear ankle thickness±SEM in naïve wild-type or RhoB KO C57BL/6 micethat received a serum transfer from arthritic K/BxN mice on day 0.

FIG. 9A provides the nucleotide (SEQ ID NO: 9) and amino acid (SEQ IDNO: 10) sequences of the light chain of 7F7. FIG. 9B provides thenucleotide (SEQ ID NO: 11) and amino acid (SEQ ID NO: 12) sequences ofthe heavy chain of 7F7. Vertical lines represent borders betweendomains. Bold—variable region (V); underlined—joining region (J);italics—diversity region (D); FWR—framework region; CDR—complementaritydetermining region.

FIG. 10A provides the nucleotide (SEQ ID NO: 13) and amino acid (SEQ IDNO: 14) sequences of the light chain of 9G5. FIG. 10B provides thenucleotide (SEQ ID NO: 15) and amino acid (SEQ ID NO: 16) sequences ofthe heavy chain of 9G5. Vertical lines represent borders betweendomains. Bold—variable region (V); underlined—joining region (J);italics—diversity region (D); FWR—framework region; CDR—complementaritydetermining region.

DETAILED DESCRIPTION OF THE INVENTION

Stable hybridomas that produce a monoclonal antibody directed againstRhoB have been difficult to generate and maintain. While attempting toobtain a hybridoma, it has been observed that the most relevanthybridomas either die or stop secreting the anti-RhoB antibody. Thisobservation led to the hypothesis that an antibody against RhoB mightinhibit antibody production in B cells. Herein, it is shown thatantibodies against RhoB can inhibit the secretion of immunoglobulinsfrom stimulated murine B cells. Further, it is shown herein thatantibodies against RhoB delay the onset and attenuate the course ofarthritis in an animal model of autoantibody-driven rheumatoid arthritis(RA). Diseases or disease symptoms that are the result of autoantibodyproduction would benefit from a therapy that blocks or attenuatesantibody production.

The administration of the anti-RhoB antibody may be similar to otherantibody-based therapies which are tolerable despite their non-targetedaspect for disease treatment. Examples include, but are not limited to,the antibody therapies anti-TNF (infliximab, adalimumab, etanercept),anti-CD20 (rituximab), and anti-BLyS (belimumab). These therapiesgenerally blunt inflammation or eradicate B cells or B cell function.For patients that poorly tolerate these therapies, the anti-RhoBantibody provides another therapeutic option.

The administration of RhoB antibodies will likely have low or notoxicity or side-effects. Notably, mice that are genetically deficientfor RhoB are normal and lack evident immune deficiencies, includingdeficiencies in B cell responses to antigen stimulation or IgG memoryformation. While RhoB deficient mice generate a normal IgG antibodyresponse, they did exhibit a mildly reduced IgM secondary response.Thus, the anti-RhoB technology appears to retard abnormal B cellfunction in the production of autoimmune antibodies, but it does notdisrupt normal B cell function after canonical antigenic challenge.Notably, RhoB is a stress response protein with a short half-life, so itis likely quickly depleted as well as functionally impaired by aspecific antibody blockade.

RhoB is an intracellular protein. Without being bound by theory, theanti-RhoB antibody may enter cells through the Fc receptor. As such,this would result in reduced toxicity or side-effects since only cellsexpressing the Fc receptor may be susceptible to anti-RhoB antibodytherapy. Similarly, toxicity should also be lower than non-targetedimmunomodulatory agents such as dexamethasone, prednisone, orthalidomide, which are used in clinic presently.

While the instant invention discloses anti-RhoB antibody therapy, otherinhibitors of RhoB (e.g., RhoB activity and/or expression) may be usedin place or in coordination with the anti-RhoB antibodies. For example,nucleic acid molecules which inhibit RhoB expression may be used such assiRNA and antisense molecules. Micro-RNA-21 has been shown to reduceRhoB expression (Sabatel et al. PLoS One (2011) 6:e16979). Additionally,RhoB peptide sequences identified herein or structurally related smallmolecules based on the peptide sequences or CDRs which interact withcorresponding epitopes on RhoB may also serve as inhibitors of RhoBactivity, particularly when coupled to appropriate delivery systems.

Antibody-mediated disruption of RhoB retards, inhibits, and/or bluntsinflammatory cellular responses that involve B cells. As mentionedabove, antibodies against RhoB can be used to alleviate diseases ordisease symptoms that are the result of autoantibody production and/orsecretion. However, specific RhoB targeted therapeutics (e.g., deliveredvia an intracellular delivery systems for macromolecules (e.g., thevariable region of an IgG molecule)) may be designed that arrest orre-direct intracellular inflammatory signals that are organized by Bcells. In this manner, the anti-RhoB therapy will work in cell typesthat contribute to chronic inflammation, such as mesenchymal cells(endothelial cells, myofibroblasts, smooth muscle cells,monocyte/macrophages) that are thought to contribute to the developmentof cardiovascular disease (CVD), cancer, diabetes or other majordiseases that may be directly or indirectly supported by an inflammatorytissue environment.

In CVD, preclinical studies have shown that RhoB is regulated by statinsand there is clinical evidence that the “non-cholesterol lowering”effect of statins can be attributed to anti-inflammatory actions. Thus,anti-RhoB therapies may be used to limit atherosclerosis or be combinedwith statins or other anti-inflammatory therapeutics as new therapeuticoptions. In other inflammatory tissue settings, anti-RhoB can alsoinhibit the inflammatory response of fibroblasts. As such, anti-RhoBtherapy can blunt fibrotic responses that contribute to tissue scarring,such as in skin, liver or heart.

With regard to diabetes, it has been shown that autoantibodies wererequired for the activation of disease causing T cells (Harbers et al.(2007) J. Clin. Invest., 117:1361-1369). Accordingly, the development ofapproaches to prevent autoantibodies from activating T cells (e.g., byreducing or inhibiting autoantibodies) would prevent or treat autoimmunedisease. Notably, it has been demonstrated that antibodies specific forCD20 can reduce the onset of diabetes by depleting a subset of B cells(Hu et al. (2007) J. Clin. Invest., 117:3857-3867). In addition todiabetes, antibody mediated treatment of other autoimmune diseases havebeen demonstrated. For example, it has been shown that antibodiesagainst the sphingosine 1-phosphate receptor reduced colitis in a mousemodel (Liao et al. (2009) FASEB J., 23:1786-96).

As stated hereinabove, the instant invention provides compositions andmethods for the inhibition, treatment, and/or prevention of autoimmunediseases and/or inflammatory diseases. In a particular embodiment, theautoimmune diseases or inflammatory diseases to be treated by themethods of the invention are those in which B-cells are implicated inthe pathophysiology and/or the symptoms of disease. Such autoimmunediseases and inflammatory disease may also be referred to as B-cellmediated autoimmune diseases or inflammatory disease. B-cells have beenimplicated in playing a role in the pathophysiology of a variety ofautoimmune or inflammatory diseases (see, e.g., Browning, J. L. (2006)Nat. Rev. Drug Discov., 5:564-576).

As used herein, the term “autoimmune disease” refers to the presence ofan autoimmune response (an immune response directed against an auto- orself-antigen) in a subject. Autoimmune diseases include diseases causedby a breakdown of self-tolerance such that the adaptive immune systemresponds to self antigens and mediates cell and tissue damage. In aparticular embodiment, autoimmune diseases are characterized as being aresult of, at least in part, a humoral immune response.

Examples of autoimmune disease include, without limitation, acutedisseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagicleukoencephalitis, Addison's disease, agammaglobulinemia, allergicasthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosingspondylitis, antibody-mediated transplantation rejection,anti-GBM/Anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmuneangioedema, autoimmune aplastic anemia, autoimmune dysautonomia,autoimmune hepatitis, autoimmune hyperlipidemia, autoimmuneimmunodeficiency, autoimmune inner ear disease (AIED), autoimmunemyocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmunethrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmuneurticaria, axonal & neuronal neuropathies, Balo disease, Behcet'sdisease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiacdisease, Chagas disease, chronic fatigue syndrome, chronic inflammatorydemyelinating polyneuropathy (CIDP), chronic recurrent multifocalostomyelitis (CRMO), Churg-Strauss syndrome, cicatricialpemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome,cold agglutinin disease, congenital heart block, coxsackie myocarditis,CREST disease, essential mixed cryoglobulinemia, demyelinatingneuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease(neuromyelitis optica), discoid lupus, Dressler's syndrome,endometriosis, eosinophilic fasciitis, erythema nodosum, experimentalallergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosingalveolitis, giant cell arteritis (temporal arteritis),glomerulonephritis, goodpasture's syndrome, granulomatosis withpolyangiitis (GPA), Graves' disease, Guillain-Barre syndrome,Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia,Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia,hypergammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgAnephropathy, IgG4-related sclerosing disease, immunoregulatorylipoproteins, inclusion body myositis, inflammatory bowel disease,insulin-dependent diabetes (type 1), interstitial cystitis, juvenilearthritis, juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome,leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneousconjunctivitis, linear IgA disease (LAD), lupus (SLE), lyme disease,Meniere's disease, microscopic polyangiitis, mixed connective tissuedisease (MCTD), monoclonal gammopathy of undetermined significance(MGUS), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis,myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's),neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromicrheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric DisordersAssociated with Streptococcus), paraneoplastic cerebellar degeneration,paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome,Parsonnage-Turner syndrome, pars planitis (peripheral uveitis),pemphigus, peripheral neuropathy, perivenous encephalomyelitis,pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, &III autoimmune polyglandular syndromes, polymyalgia rheumatic,polymyositis, postmyocardial infarction syndrome, postpericardiotomysyndrome, progesterone dermatitis, primary biliary cirrhosis, primarysclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathicpulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia,Raynauds phenomenon, reflex sympathetic dystrophy, Reiter's syndrome,relapsing polychondritis, restless legs syndrome, retroperitonealfibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidtsyndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicularautoimmunity, stiff person syndrome, subacute bacterial endocarditis(SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis,temporal arteritis/Giant cell arteritis, thrombocytopenic purpura (TTP),Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis,undifferentiated connective tissue disease (UCTD), uveitis, vasculitis,vesiculobullous dermatosis, vitiligo, Waldenstrom's macroglobulinemia(WM), and Wegener's granulomatosis (Granulomatosis with Polyangiitis(GPA)).

In a particular embodiment, the autoimmune disease is selected from thegroup consisting of rheumatoid arthritis, type 1 diabetes, systemiclupus erythematosus (lupus or SLE), myasthenia gravis, multiplesclerosis, scleroderma, Addison's Disease, bullous pemphigoid, pemphigusvulgaris, Guillain-Barr-syndrome, Sjogren syndrome, dermatomyositis,thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonalgammopathy of undetermined significance (MGUS), Waldenstrom'smacroglobulinemia (WM), chronic inflammatory demyelinatingpolyradiculoneuropathy (CIDP), Hashimoto's Encephalopathy (HE),Hashimoto's Thyroiditis, Graves' Disease, Wegener's Granulomatosis, andantibody-mediated transplantation rejection (e.g., for tissuetransplants such as renal transplant). In a particular embodiment, theautoimmune disease is type 1 diabetes, lupus, or rheumatoid arthritis.

As used herein, an “inflammatory disease” refers to a disease caused byor resulting from or resulting in inflammation. The term “inflammatorydisease” may also refer to a dysregulated inflammatory reaction thatcauses an exaggerated response by macrophages, granulocytes, and/orT-lymphocytes leading to abnormal tissue damage and cell death. In aparticular embodiment, the inflammatory disease comprises anantibody-mediated inflammatory process. An “inflammatory disease” can beeither an acute or chronic inflammatory condition and can result frominfections or non-infectious causes. Inflammatory diseases include,without limitation, atherosclerosis, arteriosclerosis, autoimmunedisorders, multiple sclerosis, systemic lupus erythematosus, polymyalgiarheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis,bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoidarthritis, inflammatory arthritis, Sjogren's Syndrome, giant cellarteritis, progressive systemic sclerosis (scleroderma), ankylosingspondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid,diabetes (e.g., Type I), myasthenia gravis, Hashimoto's thyroditis,Graves' disease, Goodpasture's disease, mixed connective tissue disease,sclerosing cholangitis, inflammatory bowel disease, Crohn's Disease,ulcerative colitis, pernicious anemia, inflammatory dermatoses, usualinterstitial pneumonitis (UIP), asbestosis, silicosis, bronchiectasis,berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamativeinterstitial pneumonia, lymphoid interstitial pneumonia, giant cellinterstitial pneumonia, cellular interstitial pneumonia, extrinsicallergic alveolitis, Wegener's granulomatosis and related forms ofangiitis (temporal arteritis and polyarteritis nodosa), inflammatorydermatoses, hepatitis, delayed-type hypersensitivity reactions (e.g.,poison ivy dermatitis), pneumonia, respiratory tract inflammation, AdultRespiratory Distress Syndrome (ARDS), encephalitis, immediatehypersensitivity reactions, asthma, hayfever, allergies, acuteanaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis,cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury),allograft rejection, host-versus-graft rejection, appendicitis,arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis,cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis,dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis,epicondylitis, epididymitis, fasciitis, fibrositis, gastritis,gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis,myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis,otitis, pancreatitis, parotitis, pericarditis, pharyngitis, pleuritis,phlebitis, pneumonitis, proctitis, prostatitis, rhinitis, salpingitis,sinusitis, stomatitis, synovitis, testitis, tonsillitis, urethritis,urocystitis, uveitis, vaginitis, vasculitis, vulvitis, andvulvovaginitis, angitis, chronic bronchitis, osteomylitis, opticneuritis, temporal arteritis, transverse myelitis, necrotizingfascilitis, and necrotizing enterocolitis. In a particular embodiment,the inflammatory disease is selected from the group consisting ofatherosclerosis, arteriosclerosis, autoimmune disorders, multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis,inflammatory arthritis, and myocarditis.

The instant invention also encompasses compositions and methods for theinhibition, treatment, and/or prevention of conditions or disordersassociated with increased levels of a certain immunoglobulin in theblood serum such as hypergammaglobulinemia or monoclonal gammopathy ofundetermined significance.

In another embodiment of the instant invention, Rho B inhibitors, e.g.,anti-RhoB antibody, are administered to a subject to treat cancerssustained by antibody secretion. In a particular embodiment, the canceris a blood tumor such as multiple myeloma. In another embodiment, thecancer is a solid tumor. Without being bound by theory, the antibodysecretion may contribute to supportive inflammatory processes.Preclinical studies show that RhoB supports tumor angiogenesis andlymphangiogenesis that are vital for malignant progression, which hasbeen demonstrated to rely upon antibody deposition in the inflammatorytumor microenvironment. Thus, anti-RhoB may be used to limit progressionof primary tumors after treatment to prevent relapses and prolongremission. Anti-RhoB therapy may also be administered to a subject totreat antibody-mediated paraneoplastic syndromes that are associatedwith certain types of cancer. Examples include, without limitation,stiff-man syndrome, opsoclonus-myoclonus (e.g., in breast cancer),peripheral encephalomyelitis, and retinopathy (e.g., in lung cancer).

The methods of the instant invention also encompass the administrationof at least one other agent for the treatment of autoimmune and/orinflammatory disease. Without being bound by theory, the administrationof anti-RhoB antibodies blunts the production of autoimmune antibodies.As such, this technology does not displace disease-specific approachesfor the treatment of the autoimmune disease.

In a particular embodiment, the method comprises administering at leastone immunosuppressant. The terms “immunosuppressant” and“immunosuppressive agent”, as used herein, include compounds orcompositions which suppress immune responses or the symptoms associatedtherewith. Immunosuppressant include, without limitation, purine analogs(e.g., azathioprine), methotrexate, cyclosporine (e.g., cyclosporin A),cyclophosphamide, leflunomide, mycophenolate (mycophenolate mofetil),steroids (e.g., glucocorticoid, corticosteroid), methylprednisone,prednisone, non-steroidal anti-inflammatory drug (NSAID), chloroquine,hydroxycloroquine, chlorambucil, CD20 antagonist (e.g., rituximab,ocrelizumab, veltuzumab or ofatumumab), abatacept, a TNF antagonist(e.g., infliximab, adalimumab, etanercept), macrolides (e.g.,pimecrolimus, tacrolimus (FK506), and sirolimus),dehydroepiandrosterone, lenalidomide, a CD40 antagonist (e.g.,anti-CD40L antibodies), abetimus sodium, BLys antagonists (e.g.,anti-BLyS (e.g., belimumab), dactinomycin, bucillamine, penicillamine,leflunomide, mercaptopurine, pyrimidine analogs (e.g., cytosinearabinoside), mizoribine, alkylating agents (e.g., nitrogen mustard,phenylalanine mustard, buslfan, and cyclophosphamide), folic acidantagonsists (e.g., aminopterin and methotrexate), antibiotics (e.g.,rapamycin, actinomycin D, mitomycin C, puramycin, and chloramphenicol),human IgG, antilymphocyte globulin (ALG), antibodies (e.g., anti-CD3(OKT3), anti-CD4 (OKT4), anti-CD5, anti-CD7, anti-IL-2 receptor (e.g.,daclizumab and basiliximab), anti-alpha/beta TCR, anti-ICAM-1,muromonab-CD3, anti-IL-12, alemtuzumab and antibodies to immunotoxins),1-methyltryptophan, and derivatives and analogs thereof. In a particularembodiment, the immunosuppressant is selected from the group consistingof methotrexate, hydroxycloroquine, CD20 antagonist (e.g., rituximab,ocrelizumab, veltuzumab or ofatumumab), abatacept, a TNF antagonist(e.g., infliximab, adalimumab, etanercept), sirolimus, and BLySantagonists (e.g., anti-BLyS (e.g., belimumab)). In a particularembodiment, the immunosuppressant is a CD20 antagonists, TNF antagonist,or BLyS antagonist.

In a particular embodiment, the methods of the instant inventioncomprise administering at least one anti-inflammatory agent. As usedherein, an “anti-inflammatory agent” refers to compounds for thetreatment of an inflammatory disease or the symptoms associatedtherewith. Anti-inflammatory agents include, without limitation,non-steroidal anti-inflammatory drugs (NSAIDs; e.g., aspirin, ibuprofen,naproxen, methyl salicylate, diflunisal, indomethacin, sulindac,diclofenac, ketoprofen, ketorolac, carprofen, fenoprofen, mefenamicacid, piroxicam, meloxicam, methotrexate, celecoxib, valdecoxib,parecoxib, etoricoxib, and nimesulide), corticosteroids (e.g.,prednisone, betamethasone, budesonide, cortisone, dexamethasone,hydrocortisone, methylprednisolone, prednisolone, tramcinolone, andfluticasone), rapamycin (see, e.g., Migita et al., Clin. Exp. Immunol.(1997) 108:199-203; Migita et al., Clin. Exp. Immunol. (1996) 104:86-91;Foroncewicz et al., Transpl. Int. (2005) 18:366-368), high densitylipoproteins (HDL) and HDL-cholesterol elevating compounds (see, e.g.,Birjmohun et al. (2007) Arterioscler. Thromb. Vasc. Biol., 27:1153-1158;Nieland et al. (2007) J. Lipid Res., 48:1832-1845; Bloedon et al. (2008)J. Lipid Res., Samaha et al. (2006) Arterioscler. Thromb. Vasc. Biol.,26:1413-1414, which discloses the use of rosiglitazone as ananti-inflammatory, Duffy et al. (2005) Curr. Opin. Cardiol.,20:301-306), rho-kinase inhibitors (see, e.g., Hu, E. (2006) Rec.Patents Cardiovasc. Drug Discov., 1:249-263), anti-malarial agents(e.g., hydroxychloroquine and chloroquine), acetaminophen,glucocorticoids, steroids, beta-agonists, anticholinergic agents, methylxanthines, gold injections (e.g., sodium aurothiomalate),sulphasalazine, penicillamine, anti-angiogenic agents, dapsone,psoralens, anti-viral agents, statins (see, e.g., Paraskevas et al.(2007) Curr. Pharm. Des., 13:3622-36; Paraskevas, K. I. (2008) Clin.Rheumatol. 27:281-287), and antibiotics (e.g., tetracyclines). In aparticular embodiment, the anti-inflammatory is a statin or high densitylipoproteins (HDL) and HDL-cholesterol elevating compound.

In accordance with another aspect of the instant invention, RhoBpeptides are provided. In a particular embodiment, the RhoB peptidecomprises at least 10 consecutive amino acids of SEQ ID NO: 3. In aparticular embodiment, the RhoB peptide comprises the C-terminal half(98 amino acids) of RhoB. In a particular embodiment, the RhoB peptideis selected from the group consisting ofVANKKDLRSDEHVRTELARMKQEPVRTDDGRAMAVRIQAYDYLECSAKTKEGVREVFETATRAALQKRYGSQNGCINCCKVL (SEQ ID NO: 5),KDLRSDEHVRTELARMKQEPVRTDDGRAMAVRIQAYDYLECSAKTKEGVREVFETAT RAAL (SEQ IDNO: 6), SDEHVRTELARMKQEPVRTDDGRAMAVRIQAYDYLECSAKTKEGVREVFETATRAALQKRYGSQNGCINCCKVL (SEQ ID NO: 7), DDGRAMAVRIQAY (SEQ ID NO: 2),RTDDGRAMAVRIQAYDYLE (SEQ ID NO: 1), and AVRIQAYDYLE (SEQ ID NO: 8) (see,e.g., FIG. 5). The RhoB peptides may be longer or shorter than the aboveidentified sequences by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more aminoacids, particularly 1, 2, 3, 4, or 5 amino acids, at the N-terminusand/or C-terminus of the peptide. In another embodiment, the peptides ofthe instant invention have at least 90%, 95%, 97%, 99%, or 100% homologyor identity with SEQ ID NO: 3 (or SEQ ID NOs: 1, 2, 5-8).

The peptides of the present invention may be prepared in a variety ofways, according to known methods. The peptides of the instant inventionmay be made by chemical peptide synthesis (e.g., solid phase synthesis).The availability of nucleic acid molecules encoding the peptide alsoenables production of the protein using in vitro expression methods andcell-free expression systems known in the art. In vitro transcriptionand translation systems are commercially available, e.g., from PromegaBiotech (Madison, Wis.) or Gibco-BRL (Gaithersburg, Md.). The peptidesmay also be produced by expression in a suitable prokaryotic oreukaryotic system. For example, part or all of a DNA molecule encodingfor the peptide may be inserted into a plasmid vector adapted forexpression in a bacterial cell, such as E. coli. Such vectors comprisethe regulatory elements necessary for expression of the DNA in the hostcell positioned in such a manner as to permit expression of the DNA inthe host cell. Such regulatory elements required for expression includepromoter sequences, transcription initiation sequences and, optionally,enhancer sequences. The peptides produced by gene expression in arecombinant prokaryotic or eukaryotic system may be purified accordingto methods known in the art.

The peptides of the invention, prepared by the aforementioned methods,may be analyzed according to standard procedures. For example, suchprotein may be subjected to amino acid sequence analysis, according toknown methods.

The peptides of the instant invention may be conjugated to a carrierprotein (e.g., a macromolecular carrier). For example, the peptides maybe used for in vivo immunization purposes. While animals may beimmunized with free peptide, anti-peptide antibody titer may be boostedby coupling the peptide to a carrier. Examples of carriers include,without limitation, KLH (keyhole limpet hemocyanin), GST(glutathione-S-transferase), BSA (bovine serum albumin), cBSA(cationized bovine serum albumin), OVA (ovalbumin), LPH (limuluspolyphenus hemocyanin), and TT (tetanus toxoid).

The instant invention also encompasses antibodies or antibody fragmentswhich are immunologically specific for RhoB (e.g., SEQ ID NO: 3). Theinstant invention also encompasses antibodies or antibody fragmentswhich are immunologically specific for amino acid sequences as set forthabove. In a particular embodiment, the peptide has at least 90%, 95%,97%, 99%, or 100% homology or identity with SEQ ID NOs: 1, 2, 5, 6, 7,or 8. The peptides may be longer or shorter than the above identifiedsequences by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids,particularly 1, 2, 3, 4, or 5 amino acids, at the N-terminus and/orC-terminus of the peptide. In another embodiment, the peptides of theinstant invention have at least 90%, 95%, 97%, 99%, or 100% homology oridentity with SEQ ID NO: 3.

The antibody molecules of the invention may be prepared using a varietyof methods known in the art. Polyclonal and monoclonal antibodies may beprepared as described in Current Protocols in Molecular Biology, Ausubelet al. eds. Antibodies may be prepared by chemical cross-linking, hybridhybridoma techniques and by expression of recombinant antibody fragmentsexpressed in host cells, such as bacteria or yeast cells.

In a particular embodiment, the antibody or antibody fragment isimmunologically specific for SEQ ID NO: 1 or SEQ ID NO: 8. In aparticular embodiment, the antibody is a monoclonal antibody, a pair ofantibodies, or a group of antibodies. In a particular embodiment, theantibody is a monoclonal antibody comprising SEQ ID NOs: 10 and 12. In aparticular embodiment, the antibody is a monoclonal antibody comprisingSEQ ID NOs: 14 and 16.

The antibody may be a naturally occurring antibody or may be a syntheticor modified antibody (e.g., a recombinantly generated antibody; achimeric antibody; a bispecific antibody; a humanized antibody; acamelid antibody; and the like). The antibody may comprise at least onepurification tag. In a particular embodiment, the framework antibody isan antibody fragment. Antibody fragments include, without limitation,immunoglobulin fragments including, without limitation: single domain(Dab; e.g., single variable light or heavy chain domain), Fab, Fab′,F(ab′)₂, and F(v); and fusions (e.g., via a linker) of theseimmunoglobulin fragments including, without limitation: scFv, scFv₂,scFv-Fc, minibody, diabody, triabody, and tetrabody. The antibody mayalso be a protein (e.g., a fusion protein) comprising at least oneantibody or antibody fragment. In a particular embodiment of the instantinvention, the antibody comprises an Fc region.

The antibody and antibody fragment of the instant invention may compriseat least one domain from the anti-RhoB monoclonal antibodies 7F7 and9G5. For example, the antibody or antibody fragment may comprise atleast one, two, three, four, five, or all six CDR domains the anti-RhoBmonoclonal antibodies 7F7 and 9G5 (see FIGS. 9 and 10). In a particularembodiment, the antibody or antibody fragment comprises at least one orboth of the CDR3 domains. In a particular embodiment, the domains of theantibody or antibody fragment have at least 90%, 95%, 97%, 99%, or 100%homology or identity with the domains present in the anti-RhoBmonoclonal antibody 7F7 or 9G5. The domains may be longer or shorterthan the domains depicted in FIGS. 9 and 10 by about 1, 2, 3, 4, or 5,amino acids, particularly 1 or 2 amino acids, at the N-terminus and/orC-terminus of the domain.

The antibody may also be a synthetic protein which mimics animmunoglobulin. Examples include, without limitation, Affibody®molecules (Affibody, Bromma, Sweden), darpins (designed ankyrin repeatproteins; Kawe et al. (2006) J. Biol. Chem., 281:40252-40263), andpeptabodies (Terskikh et al. (1997) PNAS 94:1663-1668).

The antibodies of the instant invention may be further modified. Forexample, the antibodies may be humanized. In a particular embodiment,the hybrid antibodies (or a portion thereof) are inserted into thebackbone of an antibody or antibody fragment construct. For example, thevariable light domain and/or variable heavy domain of the antibodies ofthe instant invention may be inserted into another antibody construct.Methods for recombinantly producing antibodies are well-known in theart. Indeed, commercial vectors for certain antibody and antibodyfragment constructs are available.

The antibodies of the instant invention may also be conjugated/linked toother components. For example, the antibodies may be operably linked(e.g., covalently linked, optionally, through a linker) to at least onedetectable agent, imaging agent, contrast agent, immunosuppressant, oranti-inflammatory agent. The antibodies of the instant invention mayalso comprise at least one purification tag (e.g., a His-tag).

Compositions comprising the RhoB inhibitors or antibodies are alsoencompassed by the instant invention. In a particular embodiment, thecomposition comprises at least one antibody or antibody fragment of theinstant invention and at least one pharmaceutically acceptable carrier.

The antibody molecules of the invention may be prepared using a varietyof methods known in the art. Antibodies may be prepared by chemicalcross-linking, hybrid hybridoma techniques and by expression ofrecombinant antibody or antibody fragments expressed in host cells, suchas mammalian cells, bacteria or yeast cells. In one embodiment of theinvention, the antibody molecules are produced by expression ofrecombinant antibody or antibody fragments in host cells. The nucleicacid molecules encoding the antibody may be inserted into expressionvectors and introduced into host cells. The resulting antibody moleculesare then isolated and purified from the expression system. Theantibodies optionally comprise a purification tag by which the antibodycan be purified.

The purity of the antibody molecules of the invention may be assessedusing standard methods known to those of skill in the art, including,but not limited to, ELISA, immunohistochemistry, ion-exchangechromatography, affinity chromatography, immobilized metal affinitychromatography (IMAC), size exclusion chromatography, polyacrylamide gelelectrophoresis (PAGE), western blotting, surface plasmon resonance andmass spectroscopy.

The instant invention also encompasses hybridomas that secretemonoclonal RhoB antibodies. Presently, RhoB hybridomas are—onaverage—growing and produce lower quantities of antibody compared toother hybridomas. Several approaches may be taken to circumvent thispossible limitation. For example, the nucleotide sequence of theanti-RhoB antibody may be cloned from the hybridomas and then anti-RhoBantibodies may be produced through molecular biological approaches. Inanother embodiment, RhoB-independent secreting hybridomas may bedeveloped or hybridoma culture conditions may be modified to maximizeantibody production.

The instant invention also encompasses methods for identifying smallmolecule or other molecular entities such as small nucleic acids,peptides, carbohydrates, and the like that are RhoB inhibitors. In aparticular embodiment, the RhoB antibodies of the instant invention orfragments thereof (particularly the CDR regions) or correspondingepitopes may be used to design RhoB inhibitors with similar biologicactivity.

Definitions

A “therapeutically effective amount” of a compound or a pharmaceuticalcomposition refers to an amount effective to prevent, inhibit, treat, orlessen the symptoms of a particular disorder or disease. The treatmentof an inflammatory disorder herein may refer to curing, relieving,and/or preventing the inflammatory disorder, the symptom of it, or thepredisposition towards it.

“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, excipient,auxilliary agent or vehicle with which an active agent of the presentinvention is administered. Pharmaceutically acceptable carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water or aqueous saline solutionsand aqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described, for example, in “Remington's PharmaceuticalSciences” by E. W. Martin.

An “antibody” or “antibody molecule” is any immunoglobulin, includingantibodies and fragments thereof, that binds to a specific antigen. Asused herein, antibody or antibody molecule contemplates intactimmunoglobulin molecules, immunologically active portions of animmunoglobulin molecule, and fusions of immunologically active portionsof an immunoglobulin molecule.

As used herein, the term “immunologically specific” refers toproteins/polypeptides, particularly antibodies, that bind to one or moreepitopes of a protein or compound of interest, but which do notsubstantially recognize and bind other molecules in a sample containinga mixed population of antigenic biological molecules.

As used herein, the term “prevent” refers to the prophylactic treatmentof a subject who is at risk of developing a condition resulting in adecrease in the probability that the subject will develop the condition.

The term “treat” as used herein refers to any type of treatment thatimparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the condition, etc.

As used herein, the terms “host,” “subject,” and “patient” refer to anyanimal, including humans.

The phrase “small, interfering RNA (siRNA)” refers to a short (typicallyless than 30 nucleotides long, particularly 12-30 or 20-25 nucleotidesin length) double stranded RNA molecule. Typically, the siRNA modulatesthe expression of a gene to which the siRNA is targeted. Methods ofidentifying and synthesizing siRNA molecules are known in the art (see,e.g., Ausubel et al. (2006) Current Protocols in Molecular Biology, JohnWiley and Sons, Inc). As used herein, the term siRNA may include shorthairpin RNA molecules (shRNA). Typically, shRNA molecules consist ofshort complementary sequences separated by a small loop sequence whereinone of the sequences is complimentary to the gene target. shRNAmolecules are typically processed into an siRNA within the cell byendonucleases. Exemplary modifications to siRNA molecules are providedin U.S. Application Publication No. 20050032733. Expression vectors forthe expression of siRNA molecules preferably employ a strong promoterwhich may be constitutive or regulated. Such promoters are well known inthe art and include, but are not limited to, RNA polymerase IIpromoters, the T7 RNA polymerase promoter, and the RNA polymerase IIIpromoters U6 and H1 (see, e.g., Myslinski et al. (2001) Nucl. AcidsRes., 29:2502 09).

“Antisense nucleic acid molecules” or “antisense oligonucleotides”include nucleic acid molecules (e.g., single stranded molecules) whichare targeted (complementary) to a chosen sequence (e.g., to translationinitiation sites and/or splice sites) to inhibit the expression of aprotein of interest. Such antisense molecules are typically betweenabout 15 and about 50 nucleotides in length, more particularly betweenabout 15 and about 30 nucleotides, and often span the translationalstart site of mRNA molecules. Antisense constructs may also be generatedwhich contain the entire sequence of the target nucleic acid molecule inreverse orientation. Antisense oligonucleotides targeted to any knownnucleotide sequence can be prepared by oligonucleotide synthesisaccording to standard methods.

Therapies and Compositions for the Treatment of Autoimmune andInflammatory Diseases

As stated hereinabove, the present invention encompasses compositionscomprising at least one anti-RhoB antibody (including fragments thereof)and at least one pharmaceutically acceptable carrier. The compositionmay further comprise at least one other anti-inflammatory agent and/orat least one immunosuppressive agent. Alternatively, at least one otheranti-inflammatory agent and/or at least one immunosuppressive agent maybe contained within a separate composition(s) with at least onepharmaceutically acceptable carrier. The composition(s) comprising atleast one anti-RhoB antibody and the composition(s) comprising at leastone other anti-inflammatory agent and/or at least one immunosuppressiveagent may be contained within a kit. Such composition(s) may beadministered, in a therapeutically effective amount, to a patient inneed thereof for the treatment of an inflammatory or autoimmune disease.In a particular embodiment, the patient is monitored at least once forthe inflammatory or autoimmune disease after administration of thecompositions of the instant invention to monitor the treatment of theinflammatory or autoimmune disease (e.g., in the case of rheumatoidarthritis, joint (e.g., hand joint) pain and/or stiffness; presence ofrheumatoid nodules; and/or presence of rheumatoid factor or rheumatoidfactor antibodies in the blood).

The compositions of the present invention can be administered by anysuitable route, for example, by injection (e.g., for local or systemicadministration), intravenous, oral, pulmonary, nasal or other modes ofadministration. In general, the pharmaceutically acceptable carrier ofthe composition is selected from the group of diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. The compositionscan include diluents of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; and additives such as detergents andsolubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol). The compositions can also be incorporated into particulatepreparations of polymeric compounds such as polylactic acid,polyglycolic acid, etc., or into liposomes or nanoparticles. Suchcompositions may influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of components of apharmaceutical composition of the present invention. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated byreference. The pharmaceutical composition of the present invention canbe prepared, for example, in liquid form, or can be in dried powder form(e.g., lyophilized).

In yet another embodiment, the pharmaceutical compositions of thepresent invention can be delivered in a controlled release system, suchas using an intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In aparticular embodiment, a pump may be used (see Langer, supra; Sefton,CRC Crit. Ref. Biomed. Eng. (1987) 14:201; Buchwald et al., Surgery(1980) 88:507; Saudek et al., N. Engl. J. Med. (1989) 321:574). Inanother embodiment, polymeric materials may be employed (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Press:Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley: New York (1984);Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. (1983) 23:61;see also Levy et al., Science (1985) 228:190; During et al., Ann.Neurol. (1989) 25:351; Howard et al., J. Neurosurg. (1989) 71:105). Inyet another embodiment, a controlled release system can be placed inproximity of the target tissues of the animal, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, (1984) vol. 2, pp. 115-138).In particular, a controlled release device can be introduced into ananimal in proximity to the site of inappropriate inflammation. Othercontrolled release systems are discussed in the review by Langer(Science (1990) 249:1527-1533).

The methods of the instant invention may further comprise theadministration of at least one other therapeutic method for thetreatment of the autoimmune disease or inflammatory disease. Forexample, in the treatment of an autoimmune disease, the anti-RhoBantibody may be co-administered with radiation of the subject's lymphnodes or with plasmapheresis.

In yet another embodiment, the present invention encompassescompositions comprising at least one RhoB sequence peptide (includingsequences thereof) and at least one pharmaceutically acceptable carrier.The composition may further comprise other agents (e.g., at least oneother anti-inflammatory agent and/or at least one immunosuppressiveagent) or be included in a kit with another composition, as describedhereinabove for the anti-RhoB antibodies. The compositions may bedelivered to a subject (e.g., therapeutic methods) as describedhereinabove for the anti-RhoB antibodies.

The following examples are provided to illustrate various embodiments ofthe present invention. The examples are not intended to limit theinvention in any way.

Example 1

RhoB-knockout mice were immunized with RhoB-peptide-KLH or KLH (keyholelimpet hemocyanin). Specifically, at Day 0, RhoB-KO mice were injectedwith RhoB-peptide-KLH or KLH in complete Freund's adjuvant (CFA). At Day14, a booster injection was given with RhoB-peptide-KLH or KLH inincomplete Freund's adjuvant (IFA). Lastly, a second booster injectionwas administered at Day 29 with RhoB-peptide-KLH or KLH in phosphatebuffered saline (PBS). Bleeds were obtained at Day 10 and Day 24 andserum was harvested at Day 32.

K/BxN TCR transgenic mice express a TCR reactive to a self-peptidederived from the glucose-6-phosphate isomerase (GPI), presented by theMHC class II molecule A^(g7) (Korganow et al. (1999) Immunity,10:451-461; Kouskoff et al. (1996) Cell, 87:811-822; Matsumoto et al.(1999) Science, 286:1732-1735). K/BxN mice spontaneously develop a veryaggressive form of arthritis at 4 to 5 weeks of age. The arthritis ofthe K/BxN mice mimics arthritis in humans in that it is chronic,progressive, symmetrical, and exhibits the same histological features ofhuman arthritis. The arthritis experienced by K/BxN mice is jointspecific and allows for the scoring of the arthritis by calipermeasurement of ankle thickness (Korganow et al. (1999) Immunity,10:451-461; Ji et al. (2001) J. Exp. Med., 194:321-330).

K/BxN mice (5 mice per group) were treated with 1) saline, 2) anti-KLHserum, or 3) anti-RhoB-peptide serum. Specifically, serum (200 μl) wasadministered i.p. to 21 day old mice. Mean ankle thickness was measuredover time as an indicator arthritis. As seen in FIG. 1, RhoB anti-seruminhibits arthritis.

K/BxN mice produce arthritogenic Abs directed against GPI, which developat high titers because of the preferential help that B cells expressingGPI-specific immunoglobulins receive from GPI-reactive T cellsdisplaying the transgene-encoded TCR. As above, K/BxN mice (5 mice pergroup) were treated with a total of 200 μl (100 μl of serum mixed with100 μl saline) (i.p.) of 1) saline, 2) anti-KLH serum, or 3)anti-RhoB-peptide serum. As seen in FIGS. 2A and 2B, the serum of K/BxNmice administered with RhoB anti-serum had reduced levels of serumanti-GPI Ig (as determined by enzyme-linked immunosorbent assay (ELISA))compared to K/BxN mice administered with KLH anti-serum or carrier aloneand reduced numbers of anti-GPI antibody secreting cells (as determinedby enzyme-linked immunosorbent spot (ELISPOT)) compared to K/BxN miceadministered with KLH anti-serum or carrier alone.

In addition to the above, it was also determined whether RhoB anti-serumaffected other cytokines in K/BxN mice. The administration of RhoBanti-serum to K/BxN mice did not significantly modulate the levels ofIFNγ, INFα, IL-6, IL-10, MCP-1, MIP-1α, MIP-1β, or RANTES compared toK/BxN mice administered with KLH anti-serum or carrier alone.

Splenocytes were isolated from the mice and B cells were fused withimmortalized myeloma cells (Sp2/0) to generate hybridomas. 48 sampleswere tested. 7 yielded strong positives to Peptide 1(RTDDGRAMAVRIQAYDYLE; SEQ ID NO: 1; amino acids 140-158 of human RhoB(GenBank Accession No. CAA29968)) and 5 yielded positives to Peptide 1and Peptide 2 (DDGRAMAVRIQAY; SEQ ID NO: 2; amino acids 142-154 of humanRhoB (GenBank Accession No. CAA29968)).

FIG. 3 provides the amino acid sequence of human RhoB. Peptide 1 isunderlined. Mice vaccinated with a peptide antigen encompassing thissequence were divided into two sets of antibodies. These two sets aredefined by slightly different but overlapping epitopes: binding of oneset of antibodies may be affected by Y156 phosphorylation, but the otherset of antibodies would not likely be (see above results distinguishingbetween Peptide 1 and Peptide 2, which lacks the tyrosine at 156). Bothsets of antibodies specifically recognize full-length RhoB protein, butonly one blocked antibody secretion by B cells in tissue culture or inanimals.

FIG. 4 provides the results of an ELISA experiment where an anti-RhoBhybridoma supernatant (Black) is demonstrated to suppress antibodysecretion by LPS-treated mouse B cells: compare the baseline(unactivated; diamond), activated red line (square), and suppressed line(triangle). The X line is a non-specific control (IDO antibody) thatdoes not suppress activation. The other lines represent supernatantsobtained from anti-RhoB hybridoma subclones out of the originalhybridoma, showing intermediate levels of suppression. Propidium iodide(PI) staining demonstrated that the B cells did proliferate in responseto LPS. An analysis using an IL6 bead array showed that the anti-RhoBhybridomas were not secreting IL6.

Example 2

K/BxN mice were treated with 500 μg of anti-RhoB monoclonal antibodies9G5 or 7F7 or control Ig before the onset of arthritis (21 days of age).FIG. 6A shows that both anti-RhoB monoclonal antibodies 9G5 and 7F7inhibited arthritis as indicated by rear ankle thickness. FIGS. 6B and6C show that the anti-RhoB monoclonal antibodies also inhibitautoantibody production as anti-GPI autoantibody titers were measured byELISA (FIG. 6B) and anti-GPI antibody secreting cells (ASCs) weremeasured by ELISpot assay (FIG. 6C).

K/BxN mice were also treated with 500 μg of anti-RhoB monoclonalantibodies 9G5 or 7F7 or control Ig after the onset of arthritis (4weeks of age). As seen in FIG. 7, anti-RhoB monoclonal antibodies 9G5and 7F7 inhibited the progression of arthritis, as determined by rearankle thickness.

In addition to the above, it was also determined whether anti-RhoBmonoclonal antibodies affected other cytokines in K/BxN mice. K/BxN micewere treated with 500 μg 7F7 or control Ig at 21 days of age. Cells fromthe joint draining lymph nodes were harvested at 6 weeks of age andcultured overnight in PMA (50 ng/ml) with ionomycin (500 ng/ml).Cytokines were measured in culture supernatants by cytometric beadarray. The administration of the anti-RhoB monoclonal antibody 7F7 toK/BxN mice did not significantly modulate the levels of the inflammatorycytokines IFNγ, INFα, IL-17, IL-10, MCP-1, MIP-1α, MIP-1β, and RANTES orB-cell related cytokines IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13compared to K/BxN mice administered with control Ig.

Arthritic RhoB knockout (KO) (RhoB KO KRN.g7) mice were generated bycrossing onto the KRN.g7 background. KRN B6.g7 mice are C57BL/6 micethat express both the KRN TCR tg and the IAg7 MHC Class II moleculenecessary for KRN T cell activation, but lack the rest of theNOD-associated genes (Kouskoff et al. (1996) Cell 87:811-822). FIG. 8Ashows that RhoB KO mice had reduced arthritis compared to KRN.g7 mice,as determined by rear ankle thickness. FIG. 8B shows that serum fromRhoB KO KRN.g7 mice was also unable to induce arthritis when transferredto naïve recipients. Specifically, serum from KRN B6.g7 or RhoB KO KRNB6.g7 mice was adoptively transferred into naïve C57BL/6 mice on day 0.However, FIG. 8C shows that arthritis can be induced in RhoB ko micewhen arthritogenic K/BxN serum is adoptively transferred. Serum fromarthritic K/BxN mice was adoptively transferred into naïve wt or RhoB KOC57BL/6 mice on day 0. Notably, the observed arthritis was more severeand of a longer duration with the RhoB KO mice. Without being bound bytheory, the observed increased severity in arthritis in RhoB KO mice maybe due to the inability of the mice to clear autoantibody. Indeed,anti-GPI autoantibody titers were moderately increased in RhoB KO KRN.g7mice compared to KRN.g7 mice, but the number of anti-GPI antibodysecreting cells (ASCs) were similar between the two mice.

Additionally, it was also determined whether cytokines were affected inRhoB KO mice. Cells from the joint draining lymph nodes were harvestedat 6 weeks of age and cultured overnight in PMA (50 ng/ml) withionomycin (500 ng/ml). Cytokines were measured in culture supernatantsby cytometric bead array. When compared to KRN.g7 mice, RhoB KO KRN.g7mice did not have significantly modulated levels of the inflammatorycytokines IFNγ, INFα, IL-17, IL-10, and MCP-1 (although RANTES, MIP-1α,and MIP-1β trended slightly lower in RhoB KO KRN.g7 mice) or B-cellrelated cytokines IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. Withoutbeing bound by theory, the similarity in the mouse phenotypes of RhoB KOmice compared to mice administered an anti-RhoB antibody is furtherevidence that anti-RhoB antibodies exert their activity through theirinteraction with RhoB.

RhoB KO C57BL/6 mice also possessed normal lymphoid populations.Specifically, the percentage of lymphoid populations in bone marrow,thymus, spleen, lymph nodes, and peritoneal cavity from wild-type andRhoB KO C57BL/6 mice were measured by flow cytometry. Serum Ig levelsfrom wild-type and RhoB KO C57BL/6 mice were also measured by ELISA.Notably, no significant difference in lymphoid populations or serum Iglevels (IgM, IgG1, IgG2b, IgG2c, and IgG3) was observed betweenwild-type and RhoB KO C57BL/6 mice. RhoB−/−, RhoB+/−, or RhoB+/+C57BL/6mice were also immunized with 100 μg NP-KLH in alum on day 0. Serumsamples were taken on days 0, 5, 14, and 21 and analyzed for anti-NP IgMor IgG by ELISA. The RhoB KO mice exhibited a normal response toimmunization.

Several publications and patent documents are cited in the foregoingspecification in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of these citationsis incorporated by reference herein.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. (canceled)
 2. A composition comprising an isolated anti-RhoB antibodyor fragment thereof, wherein said antibody or fragment thereof comprisesone, two, three, four, five, or all six complementarity-determiningregions (CDRs) of an anti-RhoB antibody comprising SEQ ID NOs: 10 and 12or SEQ ID NOs: 14 and 16, and at least one pharmaceutically acceptablecarrier.
 3. The composition according to claim 2, wherein the isolatedanti-RhoB antibody or fragment thereof comprises SEQ ID NOs: 10 and 12.4. The composition according to claim 2, wherein the isolated RhoBantibody comprises SEQ ID NOs: 14 and
 16. 5. The composition accordingto claim 2, wherein the isolated anti-RhoB antibody or fragment thereofis a chimeric, bispecific, and/or humanized antibody.
 6. The compositionaccording to claim 2, wherein the isolated anti-RhoB antibody orfragment thereof is a dAb, Fab, Fab′, F(ab′)₂, Fv, scFv, scFv₂, scFv-Fc,minibody, diabody, triabody, or tetrabody.
 7. The composition accordingto claim 2, wherein the isolated anti-RhoB antibody or fragment thereofis linked to at least one detectable agent, imaging agent, contrastagent, immunosuppressant, or anti-inflammatory agent.
 8. The compositionaccording to claim 2, wherein said composition further comprises atleast one anti-inflammatory agent.
 9. The composition according to claim2, wherein said composition further comprises at least oneimmunosuppressant.